Microfluidic model for in vitro acute Toxoplasma gondii infection and transendothelial migration

The protozoan parasite Toxoplasma gondii (T. gondii) causes one of the most common human zoonotic diseases and infects approximately one-third of the global population. T. gondii infects nearly every cell type and causes severe symptoms in susceptible populations. In previous laboratory animal studies, T. gondii movement and transmission were not analyzed in real time. In a three-dimensional (3D) microfluidic assay, we successfully supported the complex lytic cycle of T. gondii in situ by generating a stable microvasculature. The physiology of the T. gondii-infected microvasculature was monitored in order to investigate the growth, paracellular and transcellular migration, and transmission of T. gondii, as well as the efficacy of T. gondii drugs.

www.nature.com/scientificreports/ can more easily infect EC cells, but cannot migrate across them. The models were incapable of simulating the migration of tachyzoites across the EC monolayer into the extracellular matrix (ECM) and the complex interaction with neighboring cells. Other models with cerebral organoid 27 , organoid derived monolayers 28 , EC beads 29 , and incorporated ECMs (e.g., collagen and Matrigel) [30][31][32] have demonstrated T.gondii's interaction with adjacent environmental factors. However, they located T.gondii on EC monolayer and made the pathogens penetrate vertically, necessitating the use of a confocal microscope for observation. In this study, we hope to direct pathogen movement and penetration in a horizontal direction through a vertically cultured EC monolayer, thereby providing a clear investigation on the complex T. gondii lytic cycle. We used hydrogel incorporating microfluidic platform reconstituting blood vessels to study angiogenesis [33][34][35] , vasculogenesis 36,37 , drug permeability 26 , and the transendothelial migration of immune 38 and cancer cells 39 . Protocols were revised to examine tachyzoite migration through the EC monolayer into the ECM, and toward the neurons in order to investigate infection, proliferation, and migration of T. gondii, interacting with host cells and the surrounding microenvironment. under a microscope, a lytic cycle of the tachyzoite interacting EC monolayer, ECM and neurons was clearly observed in quantitative manner.

Materials and methods
Preparation of GFP-expressing T. gondii. The transgenic green fluorescent protein (GFP)-expressing T.
gondii strain was created by Prof. Yoshifumi Nishikawa (National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Japan). GFP-positive tachyzoites were kindly provided by Dr. Young-Ha Lee (Chungnam National University School of Medicine, Korea). Tachyzoites was electroporated by PLK strain using transfer vector, plasmid containing DHFR-GRA5′-MCSGRA3′-GRA5′-GFP-GRA3′ fragment 40 . HeLa cells (ATCC, Manassas, VA, USA) were inoculated with GFP-positive tachyzoites at a ratio of 1:3. After 3 days, tachyzoites were harvested from infected HeLa cells and passed twice through a 25-gauge needle and a 5 μm syringe filter (Millipore, USA) to remove cellular debris and host cells. The cells were then washed with Dulbecco's phosphate-buffered saline (DPBS), centrifuged (3000×g for 5 min), and suspended in DPBS.

Preparation of EC and neuron.
Purchased human microvascular endothelial cells (hMVECs) were cultured in complete medium (Lonza, Basel, Switzerland). The hMVECs were used at passages 7 and 8, with a seeding density of 2 × 10 6 cells/ml. Neurons were isolated from the brain cortex of E15 ICR mouse euthanized by CO 2 inhalation, according to procedures 41,42 , and were then immediately seeded and cultured. All animal experiments were conducted in accordance with institutional guidelines and protocols approved by Korea University's Institutional Animal Care and Use Committee (KUIACUC-2017-71 and KUIACUC-2022-0028). The study was reported in accordance with ARRIVE guidelines. www.nature.com/scientificreports/ Preparation of the microfluidic assay. An ECM-mimicking hydrogel incorporating microfluidic assay was fabricated using soft lithography of polydimethylsiloxane (PDMS; Sylgard 184; Dow Corning, MI, USA) as described previously 43 . The chip was sealed with a cover glass by using O 2 plasma (Femto Science, Seoul, Korea). Channels were filled with poly-d-lysine (PDL; Sigma, St. Louis, MO, USA) solution and incubated at 37 °C for 2 h. The PDL solution was washed and aspirated twice with sterile deionized water (DDW) and stored in an 80 °C dry oven until the day of the experimentation. Type I collagen solution was prepared with 10× PBS, NaOH, DDW, and type I collagen (BD Biosciences, CA, USA). The solution was then injected into the hydrogel channel and gelled for 30 min in a 37 °C incubator. After gelation, cell culture medium was filled into the medium channels. Recombinant basement membrane (rBM) was formed on the ECM using a Matrigel (BD Biosciences, CA, USA) coating to form a confluent and stable EC monolayer 44 . After rBM reconstitution, hMVECs were seeded at a density of 2 × 10 6 cells/ml and cultured for 5 days to form a confluent EC monolayer (Fig. 1b).

Imaging and analysis.
Fluorescence microscopy (Zeiss, Swiss) was used to follow the infection and migration of tachyzoites. For imaging purposes, cells were fixed using 4% paraformaldehyde and permeabilized with a 0.1% Triton-X100 solution. Tachyzoites were tagged with GFP, and the cytoskeleton of ECs was stained with rhodamine and phalloidin (Invitrogen, MA, USA). 3D images were acquired with confocal microscopy (LSM700, Zeiss, Swiss).

T. gondii infection and its transmigration to the confluent three-dimensional (3D) EC monolayer.
To reproduce the T.gondii infection to EC monolayer ( Fig. 1a), we used a previously reported microfluidic model with 3D EC monolayer. As demonstrated previously, the reconstituted EC monolayer on the rBM above the type 1 collagen hydrogel was found to be 3D, confluent ( Fig. 2a-c), and of low permeability 44 . Tachyzoites were seeded at a density of 2 × 10 6 cells/ml on the EC monolayer and incubated at 37 °C for 4 h, to induce microneme protein 48 mediated adhesion. The medium in the channels was refreshed every 12 h, during which time non-adherent tachyzoites were removed. The Lytic cycle of the tachyzoites in the EC monolayer, including infection, replication, proliferation, and egress was monitored ( Fig. 2d-f). The tachyzoites proliferated in the infected ECs for 36 h before actively egressing from the host ECs and migrating through the ECM to invade the adjacent ECs. The normalized infected area increased rapidly after 36 h, indicating that the in vitro cycle (of infection to egress) was approximately 36 h, as previously reported 49 . Up to 60 h, the normalized increase rate of the infected area fraction was greater than 2.2 times per 12 h. However, the rate of area increase begins to slow at 72 h, peaks at 84 h, and then decreases at 96 h. Since the majority of the surrounding cells were damaged or dead, the egressed T. gondii was washed away by the flow when the culture medium was changed (Fig. 2e). The sequential steps of tachyzoites and ECs interaction were captured in a single image (Fig. 2f). In the case of uninfected or recently infected ECs, the junction remained intact and F-actins were evenly distributed around the nucleus ( Fig. 2f(i)). After replication, the ECs became spherical and the intercellular spaces widened (Fig. 2f(ii)). During egression, the ECs disappeared ( Fig. 2f(iii)). Some tachyzoites directly penetrate the EC monolayer during the early stages of infection (12 h after infection) (Fig. 3a(i-ii)). This penetration is clearly distinct from general mechanism, infecting ECs ( Fig. 3b(iii)), migrating into the ECM after egress (Fig. 3b(iv)), and causing partial damage of ECs (white arrows) with a gap observed (yellow arrow) (Fig. 3b(iv)). This experiment cannot determine conclusively whether tachyzoite transmigration is paracellular or intracellular. Previous studies that cultured 2D EC monolayers in petri dishes or transwells 18,20 could not make these observations.

3D migration of T. gondii from the EC monolayer co-cultured with neurons. A microfluidic pro-
tocol for the co-culture of an EC monolayer with adjacent neurons was developed to model the encysted parasites that are commonly found in the brain during chronic infection 50 . Neurons were seeded on one side of the ECM to facilitate the 3D growth of axons in the ECM. After rBM coating, ECs were seeded on the opposite side of the ECM, and after 5 days, a confluent monolayer formed (Fig. 4a). After 5 days, neurons extended axons into the ECM (Fig. 4b). After a 36-h lytic cycle, tachyzoites infected the confluent EC monolayer and moved directly into the ECM (Fig. 4c,d). Notably, co-culturing neurons did not increase the number of tachyzoites in the EC monolayer, but it did increase their migration into the ECM (Fig. 5a,b). Figure 5c,d depict the egress of tachyzoites into the ECM when neurons are cultured, as indicated by the increased number of tachyzoites near the EC monolayer at 36 h. At 60 h, tachyzoites cultured in the absence of neurons began to egress from the ECs and reach a similar population in the ECM (Fig. 5e,f). Co-culturing tachyzoites with neurons appeared to enhance their invasion into ECM, but to have no effect on the invasion-egress cycle or the number of tachyzoites on the EC monolayer. Tachyzoites preferred to migrate out of neuron co-cultured EC monolayer, to initiate cycles of duplication, proliferation, and egress within the ECs. After 48 h, the migration of tachyzoites towards the neurons slowed down. According to a previous study, tachyzoites are co-localized with neurons, transform into bradyzoites and form brain cysts 10,51 . Previous studies have reported the presence of in vivo cysts in the neuron soma of the brain 52,53 and the possibility of encephalitis 54 . In our experiments, we were unable to detect bradyzoites, tachyzoites infected neurons, or the formation of cysts (Supplementary Fig. 1). www.nature.com/scientificreports/

Effect of chemicals on T. gondii infection, egress, and transendothelial migration.
In the microfluidic assay, three chemicals were evaluated: a tyrosine kinase inhibitor (genistein) 45 , a myosin II inhibitor (blebbistatin) 55 , and a calcium ionophore (A23817) 47 (Fig. 6a-d). Each chemical was added to the medium and refreshed every 12 h until the end of experiment. The tachyzoite-infected area fraction and cluster size within infected cells were measured also every 12 h (Fig. 6e,f). It was discovered that both genistein and calcium ionophores significantly reduced the overall infection caused by tachyzoites. Only genistein, however, maintained tachyzoite proliferation within infected ECs. The ability of kinase inhibitors to block the effect of calcium ionophores on the egress of tachyzoites from host cells, such as kidney cells 55 or neutrophils 56 , has been reported in prior researches. Using a microfluidic chip, we evaluated tyrosine kinase inhibitors to prevent the spread of T. gondii for the drug candidates of previous papers on T. gondii. Our experiments confirmed that microfluidic chips could be a promising platform for drug screening with parasitic infections. The tyrosine kinase inhibitor could be a promising target for the development of anti-T. gondii therapies. Calcium ionophore A23817 is known to inhibit host cell invasion and the intracellular replication of tachyzoites 57 , in addition to enhancing the egress of tachyzoites from host ECs prior to proliferation 47,58 . Experiments confirmed that the infected area fraction and cluster size of the tachyzoites decreased drastically as a result of the calcium ionophore's strong inhibitory effect on tachyzoite replication in host cells. However, A23817 demonstrated severe toxicity in ECs, which may restrict the drug's application. It is known that blebbistatin inhibits the actomyosin motor, myosin II, which forms bipolar filaments that constitute the contractile array by interlocking with actin filaments, thereby inhibiting ionophore-induced egress 46 . Nonetheless, blebbistatin was discovered to facilitate the egress of tachyzoites www.nature.com/scientificreports/  Data are presented as averaged ± SD. Statistical significance was analyzed for panels (b) and (d-f) by one-way ANOVA with Holm-Sidak method and is indicated by asterisks as follows: *P < 0.05, **P < 0.01, ***P < 0.001. www.nature.com/scientificreports/ from their host ECs prior to complete proliferation. This finding supports the notion that myosin II also plays a role in tachyzoite egress from host cells, similar to its role in virus egress 46,59 .

Discussions
We replicated 3D tachyzoite infection of EC monolayer adjacent to neurons in a microfluidic chip. Multiple days of T. gondii lytic cycle could be successfully monitored. T.gondii was able to easily infect EC monolayer, which contained their proliferation. T.gondii egressed from the infected EC monolayer and migrated through the ECM towards neurons. The inability to demonstrate neuronal infection of tachyzoites is one of the limitations of our research. Migration of tachyzoites was enhanced by the presence of neurons on the opposite side of the ECM, possibly due to neuron-secreted substances. Physical interaction between tachyzoites and axons could not account for the enhanced migration. Throughout their invasion, tachyzoites did not align with axons. Axons grew over only half of the ECM hydrogel, not reaching the EC monolayer; however there was a dramatic increase of tachyzoites around the EC monolayer 36 h after infection (Fig. 5d). The dramatic trans-endothelial migration may be attributable to the stiff gradient of enriched neuron-secreted substances over the EC monolayer. In our studies, tachyzoites did not infect neurons directly. For the reconstitution of advanced clue for the infection, i.e. cyst formation in brain, a complex cerebral microenvironment may be required, which could be realized with cerebral organoids in future research. Through drug screening experiments (Fig. 6), the precise mechanism of anti-T. gondii drugs including A23187, Genistein and belbbistatin could be verified. A23187 was found very effective at eliminating T. gondii, but also caused previously unreported severe EC damage. The mechanism by which A23187 inhibits the egress of T. gondii appears to be completely distinct from that of genistein. The death of host cells can inhibit T. gondii proliferation, but also causes severe stromal damage. Additional in vitro experiments are required to determine the optimal concentration for pathogen suppression with minimal toxicity. The balance could be monitored using the developed model, which could be beneficial for the development of new anti-pathogenic drugs, considering effect on the microenvironmental factors. This in vitro study enables sequential real-time observations of the T. gondii lytic cycle, which was previously only confirmed in mouse model with expensive two-or multi-photon microscopy 60,61 . The developed method could be integrated with existing organoid technology as an advanced infection model with microenvironment 62 . Spatial segmentation of various microenvironmental components, including blood vessels, ECMs and neurons, was reconstituted, for the temporal application of a pathogen, T.gondii (Supplementary information).

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.